Novel mammalian catecholamine receptor genes and uses

Abstract

The present invention relates to novel mammalian catecholamine receptor proteins and genes that encode such proteins. The invention is directed toward the isolation and characterization of mammalian catecholamine receptor proteins. The invention specifically provides isolated complementary DNA copies of mRNA corresponding to rat and human homologues of a mammalian catecholamine receptor gene. Also provided are recombinant expression constructs capable of expressing the mammalian catecholamine receptor genes of the invention in cultures of transformed prokaryotic and eukaryotic cells, as well as such cultures of transformed cells that synthesize the mammalian catecholamine receptor proteins encoded therein. The invention also provides methods for screening compounds in vitro that are capable of binding to the mammalian catecholamine receptor proteins of the invention, and further characterizing the binding properties of such compounds in comparison with known catecholamine receptor agonists and antagonists. Improved methods of pharmacological screening are provided thereby.

What is claimed is:  
     
         1 . A homogeneous composition of a mammalian catecholamine receptor or derivative thereof having a molecular weight of about 39 kilodaltons and an amino acid sequence identified by SEQ ID NO: 2.  
     
     
         2 . A homogeneous composition of a mammalian catecholamine receptor or derivative thereof having a molecular weight of about 38 kilodaltons and an amino acid sequence identified by SEQ ID NO: 4.  
     
     
         3 . A cell membrane preparation comprising a mammalian catecholamine receptor or derivative thereof having a molecular weight of about 39 kilodaltons and an amino acid sequence identified by SEQ ID NO: 2.  
     
     
         4 . A cell membrane preparation comprising a mammalian catecholamine receptor or derivative thereof having a molecular weight of about 38 kilodaltons and an amino acid sequence identified by SEQ ID NO: 4.  
     
     
         5 . A recombinant cell comprising a mammalian catecholamine receptor or derivative thereof having a molecular weight of about 39 kilodaltons and an amino acid sequence identified by SEQ ID NO: 2.  
     
     
         6 . A recombinant cell comprising a mammalian catecholamine receptor or derivative thereof having a molecular weight of about 38 kilodaltons and an amino acid sequence identified by SEQ ID NO: 4.  
     
     
         7 . A method of screening a compound for binding to a mammalian catecholamine receptor in cells expressing the receptor, the method comprising the steps of: 
 (a) contacting a cell culture or cell membrane with the compound, wherein the cell culture or cell membrane comprises a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 2 or SEQ ID NO: 4; and    (b) assaying the cell culture or cell membrane to determine whether the compound binds to the mammalian catecholamine receptor.    
     
     
         8 . The method of  claim 7 , wherein the cell culture comprises cells that are transformed with a recombinant expression construct that comprises a nucleotide sequence that encodes a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 2, wherein the cells of the transformed cell culture express the receptor.  
     
     
         9 . The method of  claim 7 , wherein the cell culture comprises cells that are transformed with a recombinant expression construct that comprises a nucleotide sequence that encodes a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 4, wherein the cells of the transformed cell culture express the receptor.  
     
     
         10 . A method of  claim 7  comprising the additional step of: 
 (c) comparing binding of the compound with binding of additional compounds that are known to bind to mammalian catecholamine receptors, wherein said additional compounds comprise naturally-occurring and synthetic receptor agonists and antagonists.  
 
     
     
         11 . A method of screening a compound for competitive binding to a mammalian catecholamine receptor in cells expressing the receptor, the method comprising the following steps: 
 (a) contacting a cell culture or cell membrane with the compound, wherein the cell culture or cell membrane comprises a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 2 or SEQ ID NO: 4; and    (b) assaying the cell culture or cell membrane in the presence and in the absence of an agonist for the receptor; and    (c) determining whether the compound competes with the agonist for binding to the receptor.    
     
     
         12 . The method of  claim 11 , wherein the cell culture comprises cells that are transformed with a recombinant expression construct that comprises a nucleotide sequence that encodes a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 2, wherein the cells of the transformed cell culture express the receptor.  
     
     
         13 . The method of  claim 11 , wherein the cell culture comprises cells that are transformed with a recombinant expression construct that comprises a nucleotide sequence that encodes a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 4, wherein the cells of the transformed cell culture express the receptor.  
     
     
         14 . The method of  claim 11 , wherein the compound is detectably-labeled.  
     
     
         15 . The method of  claim 11 , wherein the receptor agonist is detectably-labeled.  
     
     
         16 . The method of  claim 11 , wherein the mammalian catecholamine receptor competitor is quantitatively characterized by assaying the transformed cell culture with varying amounts of the competitor in the presence of a detectably-labeled receptor agonist and measuring the extent of competition with receptor binding thereby.  
     
     
         17 . A method of screening a compound to determine if the compound is an inhibitor of a mammalian catecholamine receptor in cells expressing the receptor, the method comprising the following steps: 
 (a) contacting a cell culture or cell membrane with the compound, wherein the cell culture or cell membrane comprises a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 2 or SEQ ID NO: 4; and    (b) assaying the cell culture or cell membrane to determine whether the compound is capable of inhibiting catecholamine receptor binding by a receptor agonist.    
     
     
         18 . The method of  claim 17 , wherein the cell culture comprises cells that are transformed with a recombinant expression construct that comprises a nucleotide sequence that encodes a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 2, wherein the cells of the transformed cell culture express the receptor.  
     
     
         19 . The method of  claim 17 , wherein the cell culture comprises cells that are transformed with a recombinant expression construct that comprises a nucleotide sequence that encodes a mammalian catecholamine receptor having an amino acid sequence identified by SEQ ID NO: 4, wherein the cells of the transformed cell culture express the receptor.  
     
     
         20 . The method of  claim 17 , wherein the compound is detectably-labeled.  
     
     
         21 . The method of  claim 17 , wherein the receptor agonist is detectably-labeled.  
     
     
         22 . The method of  claim 17 , wherein the catecholamine receptor inhibitor is quantitatively characterized by assaying the transformed cell culture with varying amounts of the inhibitor in the presence of a detectably-labeled catecholamine receptor agonist and measuring the extent of inhibition of agonist binding thereby.